Estrogens play essential roles in reproduction and contribute to the pathophysiology of breast cancer. Synthesis of estrogens from C19-steroids is catalyzed by aromatase P450 (product of the CYP19 gene). In humans, estrogens are produced in gonads, brain, placenta, bone, adipose stromal and vascular cells. Aromatase also is upregulated in breast tumors and surrounding adipose stromal cells. Expression of aromatase in each of these tissues is controlled by unique promoters that lie upstream of tissue-specific first exons that are alternatively spliced onto a common site in exon II. Interestingly, in adipose stromal cells surrounding breast tumors, switching from the weak adipose-specific promoter (promoter 1.4) to the strong ovary-specific promoter (promoter lla) occurs. During the past four years, we have successfully utilized transgenic mice to define discrete genomic regions that mediate tissue-specific human (h)CYP19 expression in placenta, ovary and brain. In cultured human trophoblast cells, we found that syncytiotrophoblast differentiation and induction of hCYPW expression are O2-dependent, and have identified response elements and transcription factors that mediate these effects and the mechanisms for O2 regulation. In the proposed research, we will continue to define critical response elements and transcription factors that mediate placenta (promoter 1.1)-, ovary- and brain (promoter 1f)-specific hCYPW expression. Our findings also indicate that the orphan nuclear receptor, LRH-1, may play an important role in gonadal development and estrogen synthesis by the ovary during the estrous cycle and pregnancy. We, therefore, will continue to assess the importance LRH-1 and other transcription factors in the regulation of CYP19 gene expression in ovary during the estrous cycle and pregnancy and in postnatal estrogen biosynthesis. We recently discovered that hCYP19 expression in breast cancer cells is greatly induced by cytokines and inhibited by progesterone acting through the progesterone receptor (PR). In the proposed research, the mechanisms for activation of hCYPW expression in breast cancer cells by inflammatory mediators, growth factors and estrogen, and for inhibition by PR will be determined. Levels of hCYP19 and cyclooxygenase-2 mRNA in human breast tumors and cell lines will be correlated with expression of estrogen receptors, PR and PR isoforms, PR-A, PR-B and PR-C, and with markers of tumor aggressiveness. The potential inhibitory role of PR on expression of hCYP19, growth factor receptors and progression to cancer also will be assessed in mice with a mammary -specific knockout of PR. We believe that this research will provide important insight into tissue-specific regulation of aromatase in normal physiology and in pathophysiologic states.